The present invention relates to purified nucleic acids encoding a krill-derived enzymes such as proteinases, which can be a multifunctional protein, and to purified polypeptides. A protein having xe2x80x9cmultifunctional activity,xe2x80x9d is defined herein as including at least one of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity, or asialo GM1 ceramide binding activity.
Multifunctional proteins are useful for multiple purposes, including treating viral infections such as herpes outbreaks, fungal, bacterial or parasitic infections, including the primary and secondary infections of leprosy, colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, cystic fibrosis, blood clots, wounds, immune disorders including autoimmune disease, such as lupus erythematosus and multiple sclerosis, and cancer. Purified polypeptides having proteinase or multifunctional activity and purified nucleic acids encoding such polypeptides are desirable to provide pharmaceutically useful products. Other uses for proteinases are well recognized in the art and include digesting proteinaceous material for a variety of purposes including cleaning and creating improved feeds for animals or bacteriology.
Until now, sequences encoding krill-derived proteinases similar to those set forth in the Sequence Listing have not been identified. The amino acid sequence included in SEQ ID NOS:4, 5, 6, 10, 20 22 or 24 or other isoforms thereof or chimeric polypeptides thereof are examples of such enzymes. In particular, in many cases the invention is specified in terms of a reference protein sequence which is AA64-300 of SEQ ID NO:4 or a sequence differing therefrom by at least one of the residue differences found in SEQ ID NOS:5, 20, 22, or 24. In an embodiment of the invention, the reference reference sequence further has the differences relative to SEQ ID NO: 4 that are found in SEQ ID NO:8. These differences are illustrated in FIGS. 5 and 6.
One preferred embodiment of the present invention is a substantially pure nucleic acid comprising a nucleic acid encoding a polypeptide having at least about 70% homology (such as identity or similarity) to a krill-derived multifunctional protein or a reference protein, such as the polypeptide of SEQ ID NOS:4, 5, 6, 8, 10, 20, 22 or 24 and in one embodiment especially SEQ ID NOS:4, 6, 10 or 22, and more preferably, at least about 80% homology, and most preferably, at least about 90% homology. Preferably, In another embodiment embodiment, the homology is with reference to SEQ ID NOS:4, 5, 20, 22 or 24. Even more preferably, the nucleic acid comprises a nucleic acid encoding a polypeptide sharing at least about 70% amino acid identity with a reference protein or, a krill-derived multifunctional protein, and yet more preferably, at least about 80% identity, and still more preferably, at least about 90% identity. Yet still more preferably, the homology or identity is at least about 95%. These levels of homology (such as identity apply to all embodiments of the invention.
In certain preferred embodiments, the substantially pure nucleic acid comprises an engineered nucleic acid variant encoding a polypeptide differing from a reference protein or a krill-derived multifunctional protein or its isoforms by no more than about 33 amino acid substitutions. and more preferably, no more than about 20 amino acid substitutions. Preferably, the engineered substitutions cause a conservative substitution in the amino acid sequence of a reference sequence or a multifunctional protein.
Preferred substantially pure nucleic acids also include nucleic acid analogs. In certain preferred embodiments, the nucleic acid comprises the open reading frames of SEQ ID NOS:1, 2, 3, 7, 9, 19, 21 or 23 and more preferably, SEQ ID NOS:1, 3, 9 or 22. In another aspect, the nucleic acid includes base pairs 190-900 of SEQ ID NO:1, base pairs 2-556 of SEQ ID NO:2, base pairs 190-900 of SEQ ID NO:3, base pairs 215-925 of SEQ ID NO:9, base pairs 1-633 of SEQ ID NO:19, base pairs 196-906 of SEQ ID NO:21, or base pairs 215-535 of SEQ ID NO:23 (in one embodiment, 1-900 of SEQ ID NO:1, base pairs 2-556 of SEQ ID NO:2, base pairs 1-900 of SEQ ID NO:3, base pairs 2-925 of SEQ ID NO:9, base pairs 1-633 of SEQ ID NO:19, base pairs 1-906 of SEQ ID NO:21, or base pairs 2-535 of SEQ ID NO:23), or more preferably base pairs 190-900 of SEQ ID NO:1, base pairs 2-556 of SEQ ID NO:2, base pairs 1-633 of SEQ ID NO: 19 base pairs 196-906 of SEQ ID NO:21 or base pairs 215-535 of SEQ ID NO:23. The invention additionally includes vectors capable of reproducing in a cell (such as a eukaryotic or prokaryotic cell) having a nucleic acid described above as well as transformed cells having such nucleic acid. Further, the invention includes a substantially pure nucleic acid comprising a nucleic acid that hybridizes, under stringent conditions, to a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a krill-derived multifunctional protein, such as the polypeptide of SEQ ID NOS:4, 5, 6, 8, 10, 20, 22 or 24 and more preferably, SEQ ID NOS:4, 6, 10 or 22.
In one embodiment, the nucleic acid is a reference nucleic acid sequence including the open reading frame sequence of SEQ ID NO:1 (or preferably preferably the portion from the NH2-IleValGlyGlyMet sequence through to the end) or a sequence differing therefrom by at least one of the bp differences found in SEQ ID NOS:2, 19, 21 or 23 (or preferably preferably the portion from the NH2-IleValGlyGly sequence through to the end). In one embodiment, the nucleic acid includes a sequence with about 50%, about 60%, about 70%, about 80%, about 90% or about 95% identity to a reference sequence.
Another preferred embodiment is a transformed cell (such as a prokaryotic or eukaryotic cell) comprising a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a krill-derived multifunctional protein. Preferably, the transformed cell expresses one of the enzymes described herein.
Yet another preferred embodiment is a vector capable of reproducing in a cell such as a eukaryotic or prokaryotic cell, the vector comprising a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a krill-derived multifunctional protein. Preferably, the inventive vector codes for expression, intracellularly or extracellularly, of one of the multifunctional proteins described herein.
Another embodiment of the present invention is a polypeptide comprising a substantially pure isoform of a reference sequence or a krill-derived multifunctional protein or engineered variant thereof, and preferably, a polypeptide comprising SEQ ID NOS:4, 5, 6, 8, 10, 20, 22 or 24 and especially, SEQ ID NOS:4, 6, 10 or 22. In one embodiment, the isoform is a polypeptide comprising SEQ ID NOS:4, 5, 20, 22 or 24. The invention further provides a pharmaceutical composition for treating an animal comprising an effective amount of such a polypeptide together with a pharmaceutically acceptable carrier.
Yet another preferred embodiment is a method of preparing an enzyme such as a multifunctional protein, wherein the protein has at least about 70% homology to a reference sequence or a krill-derived multifunctional protein. Such method comprises
(a) transforming an appropriate eukaryotic or prokaryotic cell with an expression vector for expressing intracellularly or extracellularly a nucleic acid encoding the protein;
(b) growing the transformed cell in culture; and
(c) isolating the protein from the transformed cell or the culture medium.
Yet another preferred embodiment is a pharmaceutical composition for treating an animal comprising an effective amount of an expression vector comprising a nucleic acid encoding a multifunctional protein, and a pharmaceutically acceptable carrier. The invention further provides a pharmaceutical composition for treating an animal comprising an effective amount of a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a krill-derived multifunctional protein.